Simplified quantitation of cytotoxicity by integration of specific lysis against effector cell concentration at a constant target cell concentration and measuring the area under the curve

Abstract

Experience with the lytic unit (LU) as a measure of cytolytic efficiency has indicated that its accuracy is limited, even if expressed in a logarithmic format. A new method of quantifying cytotoxicity from effector dilution assays is proposed: the area under the curve (AUC) of the Ig ( E/ T) ratio vs. percentage cytotoxicity plot, gives an overall measure of lytic efficiency. The AUC derived from the Briggs-Haldane kinetic model is dependent on both the kinetic parameters that determine the efficiency of effector cells (the Michaelis constant K M and the catalytic constant k cat). AUC provides an index of inhibition or stimulation of lysis, independent of whether the modulation is kinetically competitive, uncompetitive or mixed. The relationship between the AUC and the LU suggests that a broad range of LU data could have the same AUC value. In practice the method may be applied to interpret simple cytotoxicity assay data, where effector cells are being used in standardised screening for modifiers of the cytolytic response. Illustrative data of LAK cytotoxicity influenced by dose of the LAK response modifiers IL-2, TGFβ, TDSF and 5-FU, show different relationships between lytic units, K M and AUC. These data also shows a wide range in the Hill coefficient and would be consistent with a cooperative effect dependent on the effector cell efficacy. This confirms that using LU as a simple measure of cytolytic efficiency could be erroneous and suggest that cytolytic response modifiers can produce a variety of kinetic changes. the AUC method, however, provides a comparative measure of efficiency in these situations, independent of mechanism.